Histone H1 Depletion Impairs Embryonic Stem CellDifferentiationYunzhe Zhang1,2., Marissa Cooke2,3., Shiraj Panjwani1,2., Kaixiang Cao1,2, Beth Krauth3, Po-Yi Ho1,2,

Magdalena Medrzycki1,2, Dawit T. Berhe2, Chenyi Pan1,2, Todd C. McDevitt2,3, Yuhong Fan1,2*

1 School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 2 The Petit Institute for Bioengineering and Bioscience, Georgia Institute

of Technology, Atlanta, Georgia, United States of America, 3 The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta,

Georgia, United States of America

Abstract

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts tocharacterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remainselusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. Toinvestigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine thedifferentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are moreresistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly,the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layersand retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation ofEBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, wediscover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparisonwith wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regionsnecessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary,we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 andchromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotencygenes.

Citation: Zhang Y, Cooke M, Panjwani S, Cao K, Krauth B, et al. (2012) Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation. PLoS Genet 8(5):e1002691. doi:10.1371/journal.pgen.1002691

Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America

Received September 2, 2011; Accepted March 21, 2012; Published May 10, 2012

Copyright: � 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work is supported by a Johnson & Johnson/GA Tech Healthcare Innovation Award (to YF and TCM), a Georgia Cancer Coalition DistinguishedScholar Award (to YF), NIH grant GM085261 (to YF), NSF EBICS Science and Technology Center (CBET-0939511), the Georgia Tech Integrative BioSystems Institute,and Georgia Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: Yuhong.fan@biology.gatech.edu

. These authors contributed equally to this work.

Introduction

Pluripotent embryonic stem cells (ESCs) can self-renew and

differentiate into diverse cell types, including lineages from all

three germ layers present in the adult organism, offering great

promise in regenerative medicine in addition to serving as a useful

system for developmental biology studies. The epigenome and

transcriptional circuitry of pluripotent stem cells have been

extensively investigated, and chromatin and epigenetic signatures

have emerged as key components in defining and regulating stem

cell pluripotency [1–4]. Recent reports have associated ESCs with

a particularly open, hyperdynamic chromatin and hyperactive

global transcription [2,5,6], and open chromatin has been

suggested as a marker for pluripotency [7,8]. However, it remains

undetermined whether higher order chromatin compaction is

required for pluripotent stem cell differentiation and how an open

chromatin state impacts stem cell function.

In eukaryotic cells, histones are the major structural proteins

that associate with DNA to form chromatin. The basic repeating

unit of chromatin is the nucleosome core particle, which consists of

an octamer of four core histones (H2A, H2B, H3 and H4)

wrapped by 146 bp of DNA [9]. Further compaction of chromatin

into higher order structures, such as a 30 nm fiber, is facilitated by

binding of H1 linker histones to DNA entry/exit points of

nucleosomes and linker DNA between nucleosomes. Reducing the

total amount of H1 in vivo leads to a relaxed chromatin structure

[10–12].

The H1 histone family is the most divergent and heterogenous

group of histones among the highly conserved family of histone

proteins. In mammals, 11 non-allelic H1 subtypes have been

identified, including five somatic H1 subtypes (H1a–e), the

replacement subtype H10, four germ cell specific H1 subtypes

(oocyte specific H1oo, and testis-specific H1t, H1t2, H1LS1) as

well as a more recently identified and distantly related subtype

H1x [13]. Although the individual depletion of each of the three

major somatic H1 subtypes, H1c, H1d and H1e, in mice does

not lead to any detectable changes in total H1 levels or obvious

phenotypes [14], deletion of H1c, H1d and H1e altogether leads

to nearly a 50% reduction of total H1 levels and embryonic

lethality with a broad phenotype [15], demonstrating that

critical levels of total H1 histones are essential for mouse

embryogenesis.

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We have previously derived wild-type (WT) and H1c/H1d/

H1e triple knockout (H1 TKO) embryonic stem cells from the

outgrowth of the inner cell masses of blastocysts attained from

intercrosses of H1 heterozygous mutants [10]. We have measured

that wild-type ESCs have an H1/nucleosome ratio of 0.46 [10], a

much lower level compared with a ratio of 0.75,0.83 from

various differentiated cell types in mouse tissues [11,15], suggesting

that ESCs have a more open chromatin structure compared with

differentiated cell types in adult tissues. H1 TKO ESCs have an

even lower H1/nucleosome ratio that is close to 0.25, equivalent

to 1 H1 per 4 nucleosomes. The compound H1 null ES cells

display chromatin decondensation in bulk chromatin [10] and an

increased nuclear size [16], offering an ideal system to test the

necessity of chromatin compaction on ESC pluripotency and

differentiation.

In the current study, we demonstrate, for the first time, that the

differentiation capacity of ESCs that lack multiple H1 subtypes is

severely impaired. We find that compound H1 null ESCs are more

resistant to spontaneous differentiation, impaired in embryoid

body differentiation, and largely blocked in neural differentiation.

Finally, we present evidence that H1 contributes to efficient

repression of the expression of pluripotency factors and partici-

pates in establishment and maintenance of epigenetic marks

necessary for silencing pluripotency genes during stem cell

differentiation and embryogenesis.

Results

Loss of H1c/H1d/H1e inhibits spontaneous ESCdifferentiation

ESCs exhibit a relatively ‘‘open’’ chromatin structure compared

with differentiated cells or lineage committed cells [8]. H1c/H1d/

H1e triple null ESCs we derived previously have a significant

reduction in total H1 protein levels which leads to further

decreased chromatin compaction [10], thus we postulated that loss

of H1c, H1d, and H1e may interfere with ESC differentiation. We

first compared the spontaneous differentiation tendency of two H1

TKO ESC lines with wild-type littermate ESC lines. Consistent

with previous observations [10], H1 TKO ESCs cultured on

mitotically inactivated mouse embryonic fibroblast (MEF) feeder

cells with media containing leukemia inhibitory factor (LIF) have

comparable growth rate to that of wild-type ESCs (data not

shown) and normal karyotypes (Figure S1). In addition, H1 TKO

ESCs expressed comparable levels of pluripotency factor OCT4

(POU5F1) (Figure 1A), and displayed a similar ESC colony

morphology to that of WT ESCs under culture conditions which

promote ESC self-renewal (Figure 1B, left panel). However, when

cultured in a feeder-free manner on gelatin-coated plates without

MEFs, the H1 TKO cells displayed higher levels of OCT4, a more

homogeneous, undifferentiated colony morphology, and a higher

growth rate than WT ESCs under the same condition (Figure 1A,

1B middle panel, and 1C). Furthermore, upon removal of LIF, the

majority of H1 TKO ESCs continued to retain high expression

levels of OCT4 (Figure 1A) as well as a tightly packed colony

morphology typical of undifferentiated ESCs (Figure 1B, right

panel) for a week. In contrast, wild-type ESCs differentiated

readily, with approximate 90% of the cells appearing to

differentiate by 2 days after LIF removal in feeder free culture,

as judged by diminishing OCT4 expression and the loss of a

compact colony morphology (Figure 1A, 1B right panel). Removal

of LIF reduced the growth of both WT and H1 TKO ESCs

(Figure 1C), consistent with LIF’s known role in promoting self-

renewal and proliferation of ESCs [17]. Collectively, these results

suggest that ESCs lacking H1c, H1d, and H1e are more refractory

to spontaneous ESC differentiation in vitro.

Loss of H1c, H1d, and H1e impairs EB differentiationTo assess whether loss of H1c, H1d and H1e impairs cellular

differentiation of any of the three germ layers, we examined the

ability of H1 TKO ESCs to form embryoid bodies (EB) using a

rotary orbital suspension culture system to induce differentiation in

vitro. We have previously shown that the rotary suspension culture

method offers improved efficiency and homogeneity of embryoid

body production compared with the common practice of forming

EB aggregates in static suspension culture [18]. During EB culture

in serum-containing media, ESCs form aggregates and differen-

tiate into cell types of all three primitive germ layers: endoderm,

mesoderm and ectoderm, offering a temporal window to

investigate specific defects in lineage differentiation. After 10 days

of culture in rotary suspension, the wild-type EBs had a distinct

outer endoderm-layer surrounding differentiated cell morpholo-

gies representing the three germ layers, including different

epithelial cell types and mesenchymal cell populations

(Figure 2A). In contrast, although H1 TKO ESCs were able to

form putative EBs, most H1 TKO EBs appeared blocked in the

differentiation process in rotary suspension culture, forming

undifferentiated masses of stem cells that lacked cavity formation

and other types of differentiated structures even after prolonged

culture in rotary suspension (up to 14 days) (Figure 2A).

Quantitative RT-PCR analyses also indicated that the expression

of differentiation markers, such as the endoderm marker, alpha-

fetoprotein (AFP), was drastically increased in WT EBs, but

significantly curbed in H1 TKO EBs (Figure 2B). The mRNA

levels of other lineage specific markers, including mesoderm

markers, such as the cardiac transcription factor Nkx2.5, and the

sarcomeric muscle marker, alpha myosin heavy chain (aMHC),

also progressively increased over time in WT EBs, but were not

detected at similar levels in H1 TKO EBs (Figure S2A).

To gain a more comprehensive view of the scope of genes

affected by linker histone H1 depletion during differentiation, we

performed quantitative PCR SuperArray analysis of wild-type and

H1 TKO cells at the start (day 0) and the end point (day 10) of

rotary suspension culture. The genes analyzed included pluripo-

Author Summary

The epigenome and chromatin play critical roles in stemcell fate determination. Linker histone H1 is a majorchromatin structural protein that facilitates higher-orderchromatin folding. By analyzing the differentiation capac-ity of embryonic stem cells (ESCs) that lack multiple H1subtypes, we find, for the first time, that H1 and higher-order chromatin compaction are required for properdifferentiation and lineage commitment of pluripotentstem cells. Triple-H1 null murine ESCs are impaired in bothspontaneous differentiation and embryoid body differen-tiation. Furthermore, triple-H1 null ESCs are compromisedin neural differentiation. Finally, we demonstrate that H1depletion leads to failure of efficient repression ofpluripotency gene expression both in embryos and inESC differentiation. We present evidence that H1 partici-pates in mediating changes of histone marks and DNAmethylation necessary for silencing pluripotency geneOct4 during stem cell differentiation and embryogenesis.This finding is important because it provides a mechanisticlink by which H1 and chromatin compaction mayparticipate in pluripotent stem cell differentiation throughrepression of pluripotency gene expression.

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tency genes as well as important developmental genes for

transcription factors and signaling molecules for all three germ

layers. WT and TKO cultures at day 0 displayed very few

differences in gene expression and their gene expression profiles

clustered most similarly in hierarchical cluster analysis (Figure 2C,

2Di, and Figure S2Bi). WT EBs differentiated as expected with

significant increases of many differentiation markers and de-

creased expression of pluripotency associated genes (Figure 2C,

Figure 1. Loss of H1c/H1d/H1e inhibits spontaneous ESC differentiation. (A) Western blot analysis of OCT4 level in WT and H1 TKO ESCscultured under indicated conditions for 2 days. (B) Phase images of WT and H1 TKO ESCs cultured either on MEF with LIF (left panel), gelatin coatedplate with LIF (middle panel), or gelatin coated plate without LIF (right panel) for 2 days. Scale bar: 100 mm. (C) Growth curves of WT and H1 TKO ESCscultured on gelatin coated plate with or without LIF. Data are presented as average 6 S.D.doi:10.1371/journal.pgen.1002691.g001

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Figure 2. H1c/H1d/H1e triple knockout ESCs are impaired in EB differentiation. (A) Hematoxylin and eosin (H&E) staining of sections of WTEBs (top panels) and H1 TKO EBs (bottom panels) at 7 days, 10 days and 14 days in rotary suspension culture. High magnification images of H&Estaining of sections of WT EB (top right) and H1 TKO EBs (bottom right) show that TKO EBs failed to cavitate. WT EBs showed more differentiated

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2Dii, and Figure S2Bii). In contrast, H1 TKO EBs exhibited very

similar gene expression signatures to those of ESCs and had less

expression changes during differentiation compared with that of

WT EBs. (Figure 2C, 2Diii, and Figure S2Biii, S2C), suggesting

that the lack of H1c, H1d and H1e leads to diminished changes of

transcriptional reprogramming during differentiation. The levels

of ectoderm markers, such as Nestin (Nes), mesoderm markers, such

as Brachyury (T) and FLT1, and endoderm markers, such as AFP

and Gata4, were all markedly less or failed to be expressed in H1

TKO EBs (Figure 2C and Figure S2C), indicating that differen-

tiation to all three germ layers was suppressed.

H1 is required for neural differentiation of embryonicstem cells

To further investigate if and when H1 impacts cell differenti-

ation in a specific lineage, we induced differentiation of H1 TKO

ESCs under a neural differentiation regimen established using all-

trans retinoic acid (RA), which is known to induce neural

differentiation in ESCs [19,20]. EBs were prepared using the

hanging-drop method, and day 4 EBs were collected and treated

with RA for additional two days followed by further differentiation

with neural differentiation media on poly-L-ornithine and laminin

(PLO+L) coated tissue culture plates (Figure 3A). By day 6+7 of

this in vitro neural differentiation scheme, neural cells were clearly

established and neurite outgrowth from EBs was seen with

neuronal cell proliferation. Neurites are enriched in cylindrical

bundles of microtubules, made primarily of b-III tubulin (TUBB3)

protein, extending from the body of all neurons, finally differen-

tiating into an axon or a dendrite [21]. However, at this time

point, neural differentiation of WT and TKO ES cells exhibited

several striking differences.

While neurite-formation was efficient in WT culture with

bundles of neurites cylindrically extending from EB to adjacent

EB, H1 TKO EBs had much less neurite outgrowth (Figure 3Bi,

3Bii). Approximately 50% of WT EBs plated for neural

differentiation formed neurites compared to only about 10% of

H1 TKO EBs forming neurites (Figure 3Bii, left panel).

Furthermore, those 10% TKO EBs that were capable of forming

neurites only produced on average 8 neurites per EB, whereas

each WT EB had on average 18 neurites (Figure 3Bii, right

panel). During in vitro neural differentiation, neurons aggregated

into mounds of cells forming neuronal clusters (Figure 3Bi; black

arrows), connected by bundles of neurites (Figure 3Bi; white

arrows), forming a network pattern. While WT cultures showed

formation of a neural network with neural clusters inter-

connected by bundles of neurites, H1 TKO cultures failed to

develop such an extensive intercellular network (Figure 3Bi, ii),

evidenced by smaller neuronal clusters with negligible inter-

connecting neurites. This was further confirmed with immuno-

fluorescence detection of TUBB3 protein expression, and

minimal TUBB3 staining was seen in H1 TKO cultures

(Figure 3Biii). It appeared that both neurite formation and

outgrowth were limited in H1 knock-out mutants, affecting the

ability of neurons to form neural networks. We also noted that

TKO cultures yielded markedly less glial cells as revealed by

much fewer GFAP positive astrocytes in comparison with WT

cultures (Figure 3Biii). Since glial cells are essential for the normal

growth and development of neurons, the near-lack of glial cells in

TKO cultures may contribute to the poor development of

TUBB3 positive neuronal cells from TKO EBs.

To examine whether the aforementioned defects of the H1

TKO cultures represent a temporary delay or a blockage in neural

differentiation, we cultured the cells for an additional 14 days

under neural differentiation conditions. As expected, the neural

marker (Nestin) and the astrocyte marker (GFAP) were efficiently

and progressively induced in WT cell cultures, and the neuronal

gene Tyrosine hydroxylase (TH) peaked at day 6+7 when neuronal

proliferation occurred (Figure 3C). In contrast, the expression

levels of neural genes were significantly curtailed in H1 TKO

cultures, suggesting the lack of progression in neural differentiation

of H1 TKO culture (Figure 3C). Furthermore, we observed that

pluripotency genes Oct4 and Nanog were expressed at higher levels

in TKO than WT throughout the differentiation process

(Figure 3C). These data suggest that H1 TKO cells are largely

blocked in neural differentiation.

Levels of H1 increase progressively during differentiationTo address the mechanisms by which H1 modulates differen-

tiation, we first examined the expression profile of linker histone

H1 subtypes during EB formation and differentiation of wild-type

ESCs. Histones from wild-type, H1 TKO ESCs and EBs were

isolated at various time points during differentiation, and the levels

of individual H1 subtype proteins as well as the H1 to nucleosome

ratio were quantified from HPLC and mass spectrometry analysis

as described previously [15,22,23]. In ESCs (day 0), H10 was

nearly undetectable in WT cells but was increased in H1 TKO

cultures as we observed previously (Figure 4A and [10]). Upon EB

differentiation, the levels of H1c, H1d and H1e and H10 in WT

cultures were all progressively increased over time, with the total

H1 to nucleosome ratio elevated nearly 40% from 0.45 for ESCs

to 0.62 for day 10 EBs (Figure 4B, 4C). Consistent with HPLC

analysis, Western blotting showed that levels of total H1 and H10

were increased (Figure S3). The cumulative increase in the protein

levels of H1c, H1d and H1e was responsible for 87% of the

increase in the total H1 levels during differention (data not shown).

Despite less abundant than H1d, H1c and H1e were significantly

increased (P,0.001), and H1e levels in differentiated EBs were

over 2-fold of that in undifferentiated ESCs (Figure 4C). The

protein levels of H1a and H1b remained constant during

differentiation, indicating that H1a and H1b were not responsible

for the increase of total H1 during ESC differentiation. Albeit

higher than that in TKO ESCs (0.25), the ratio of total H1 to

nucleosome in day 10 TKO EBs (0.36) remained lower than the

ratio in WT ESCs (0.45) (Figure 4B). The increase in the total H1

level in TKO EBs compared with ESCs was largely due to the

increase in the level of H10 (Figure 4B, 4C, and Figure S3),

indicating H10 being the major H1 subtype upregulated in the

face of deficiency of H1c, H1d, and H1e, in both ESCs and EBs.

These results show that the levels of H1c, H1d, H1e and H10 are

elevated significantly during embryonic stem cell differentiation,

morphologies with cysts forming (black arrows). (B) Quantitative RT-PCR analysis of mRNA expression levels of AFP in ESCs (day 0) and EBsthroughout 14 days of rotary suspension culture. Data were normalized over the expression level of GAPDH and are presented as average 6 S.D. (C)Hierarchical clustering analysis of qRT-PCR SuperArray gene expression profiling of ESCs (day 0) and EBs (day 10) formed from WT and H1 TKO ESCs.Red, green or black represent higher, lower, or no change in relative expression. (D) Scatter Plot analysis of gene expression comparisons of: (i) WT vs.H1 TKO ESCs (day 0); (ii) WT EBs (day 10) vs. WT ESCs (day 0); (iii) H1 TKO EBs (day 10) vs. H1 TKO ESCs (day 0). X- and y- axes are delta CTs usingGAPDH to normalize. Genes with more than 2-fold differences lie outside of the blue lines.doi:10.1371/journal.pgen.1002691.g002

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Figure 3. H1 TKO ESCs fail to undergo neural differentiation. (A) Neural differentiation scheme for ESCs. (B) Characterization of WT and H1TKO cultures on day 6+7 under neural differentiation protocol. i). Phase contrast images shows that H1 TKO mutants were unable to adequately formneurites and neural networks. Right panels: zoom-in images of the areas encircled with black rectangles. Scale bar: 100 mm (left panels) and 50 mm(right panels). ii). Left panel: Percentage of neurite-forming EBs. Numbers were averaged from 6 experiments. 80 EBs were counted per experiment.Right panel: Numbers of neurites per neurite-forming EB. Number of neurites was counted from EBs that produced neurites. 58 and 28 neurite-forming EBs from respective WT and TKO were selected and counted for neurite numbers. **: P,0.01; ****: P,0.0001. iii). Immunostaining forexpression of TUBB3 and GFAP. Nuclei were stained with Hoechst 33342. Scale bars: 50 mm (left panels) and 20 mm (right panels). Results arerepresentative of three independent experiments. (C) H1 TKO ESCs were unable to adequately repress the pluripotency genes and to efficientlyinduce the expression of neural genes. Expression levels of pluripotency genes (Oct4 and Nanog), neural marker (Nestin), neuronal marker (Tyrosine

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and that the H1 TKO EB has a total H1 level lower than the WT

ESC.

H1c/H1d/H1e is necessary for efficient transcriptionalrepression of pluripotency genes Oct4 and Nanog duringembryogenesis and ESC differentiation

The results from the aforementioned experiments suggest that

H1c/H1d/H1e triple null ESCs are less effective than WT ESCs

in repressing the expression of pluripotency genes, such as Oct4

and Nanog, during spontaneous differentiation, rotary suspension

differentiation, and neural differentiation in vitro (Figure 1A,

Figure 2C, and Figure 3C). Therefore, we next investigated if H1

contributes to stable repression of pluripotency gene expression in

vivo during embryogenesis. Oct4 is expressed in undifferentiated

cells in the preimplantation embryo, and is progressively down-

regulated in differentiating embryonal cells during gastrulation,

becoming restricted to germ cell precursors after E8.5 [24],

whereas Nanog expression is largely downregulated after E4.5 [25].

We analyzed expression of Oct4 and Nanog from E8.5 embryos,

when many of the surviving TKO embryos appeared comparable

to WT littermates. E8.5 embryos were harvested from intercrosses

of H1c/H1d/H1e triple heterozygotes and the expression levels of

Oct4 and Nanog in TKO and WT embryos were analyzed from

three litters using quantitative RT-PCR. On average, expression

levels of Oct4 and Nanog in TKO embryos were more than 4-fold of

that from WT littermate controls (Figure 5Ai, Figure S4A),

hydroxylase (TH)), astrocyte marker (GFAP) from WT and H1 TKO cultures at indicated days in differentiation cultures were determined by qRT-PCR.Data were normalized over the expression level of GAPDH and are presented as average 6 S.D.doi:10.1371/journal.pgen.1002691.g003

Figure 4. Expression profiles of linker histones in WT and H1 TKO cultures during EB differentiation. (A) Reverse-phase HPLC and MassSpectrometry (inset) analysis of histones from WT and H1 TKO ESCs. X axis: elution time; Y axis: absorbency at A214. mAU, milli-absorbency units. Insetshows the relative signal intensity of H1d and H1e mass spectral peaks in the H1d/H1e fraction collected from HPLC eluates of WT histones. (B,C) H1/nucleosome ratio of the total H1 (B) and individual H1 subtype (C) during EB formation and differentiation. Day 0, day 7 and day 10 of EB cultureswere collected and HPLC analyses as shown in (A) were performed. The ratio of total H1 (or individual H1 subtype) to nucleosome was calculated asdescribed in Materials and Methods. Values are means 6 S.D., n = 4. *: P,0.05; **: P,0.01; ***: P,0.001; ****: P,0.0001.doi:10.1371/journal.pgen.1002691.g004

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indicating that depletion of H1 impairs repression of the

expression of pluripotency factors in E8.5 embryos in vivo.

DNA methylation of cytosine nucleotide at CpG sites within

gene promoter regions contributes to stable gene silencing, and

thus is a key determinant in regulating the expression of

pluripotency genes [26], so we asked if the DNA methylation

status at the Oct4 and Nanog promoters is affected in H1 TKO

embryos. Results from bisulfite sequencing analysis demonstrated

that the extent of CpG methylation at the Oct4 promoter region

was markedly reduced in triple-H1 null embryos in comparison

with corresponding wild-type littermates (Figure 5Aii), whereas the

level of DNA methylation (percent methylation of analyzed CpGs)

at Nanog promoter did not display differences between WT and H1

TKO embryos (Figure S4B, S4C). This suggests that H1

participates in establishing and/or maintaining CpG methylation

at Oct4 promoter during embryogenesis.

To further investigate the mechanisms by which H1 regulates

pluripotency genes during ESC differentiation, we analyzed the

epigenetic profiles of the Oct4 and Nanog genes during EB

differentiation in rotary suspension culture. We demonstrated

previously that this method produces a large quantity of

homogeneous EBs that progressively differentiate [18], thus the

sequential epigenetic events can be readily followed. Expression of

Oct4 and Nanog was reduced during continuous suspension culture

for WT cultures, but remained high in TKO EB cultures

(Figure 5Bi, Figure S6A). DNA methylation analysis by bisulfite

sequencing indicated that WT EBs had an increase in the sporadic

DNA methylation at specific CpG sites throughout the Oct4

proximal promoter region at day 10 (P = 0.002 and 0.036 for the

respective R1 and R2 regions) (Figure 5Bii, iii), whereas TKO EBs

remained completely unmethylated at these sites. On the other

hand, Nanog promoter region remained unmethylated throughout

the differentiation in both WT and H1 TKO cultures (Figure

S6B).

To further investigate the effect of H1 levels in affecting

expression and DNA methylation of pluripotency genes in EB

differentiation, we generated ‘‘rescue’’ cell lines (referred to as

‘‘RES’’) by stably overexpressing exogenous H1d in the H1 TKO

cells (Figure S5A). RES cells had a H1/nucleosome ratio of 0.31

(Figure S5B), displayed a normal karyotype (Figure S5C), and

were able to differentiate into EBs with cystic structures which

were observed in WT, but not in TKO, EBs (Figure S5D). RES

EBs had elevated expression of differentiation markers, such as

AFP and Nkx2.5 (Figure S5E) and reduced expression of Oct4 and

Nanog pluripotency genes upon differentiation (Figure 5Bi and

Figure S6A), suggesting that the expression of exogenous H1d

alleviates the differentiation defects and restores the repression of

pluripotency factors in H1 TKO EBs. In addition, the percent of

methylated CpG was increased in RES EBs to a level comparable

to that of WT EBs at the same time points, suggesting that

reintroduction of H1d into the H1 TKO ESCs is able to

reestablish DNA methylation and the stable repression of the Oct4

gene in differentiating EBs (Figure 5Bii, iii).

We next analyzed the status of H1, H3K4me3, H3K9me3 and

H3K27me3 at the promoters of pluripotency genes Oct4 and Nanog

by quantitative chromatin immunoprecipitation (qChIP). Whereas

H1 occupancy at Oct4 promoter increased in WT and RES

cultures during differentiation, it remained unchanged in H1

TKO EBs (Figure 5Biv). It is interesting to note that the

occupancy of the replacement subtype, H10, at Oct4 promoter

was markedly increased in both WT and RES cultures but only

mildly elevated in H1 TKO cultures (Figure S7), suggesting that

efficient binding of H10 at Oct4 promoter may be facilitated by

sufficient amount of other somatic H1s. Furthermore, wild-type

and RES EBs displayed decreasing levels of the active histone

mark H3K4me3 accompanied with a significant increase in the

levels of the repressive histone mark H3K9me3, at promoter

regions of pluripotency genes Oct4 and Nanog upon EB differen-

tiation (Figure 5Biv and Figure S6C). In contrast, H1 TKO EBs

did not display similar or significant changes in the levels of these

histone marks at the same promoter regions (Figure 5Biv and

Figure S6C). Levels of H3K27me3, another repressive histone

mark, were significantly increased in WT cultures during

differentiation at Oct4 promoter, while such increases were not

detected at H1 TKO or RES EBs (Figure 5Biv).

These analyses suggest that the increase of H1 levels and the

changes in histone modifications, such as H3K4me3, H3K9me3

and H3K27me3, precede DNA methylation establishment in

mediating Oct4 gene silencing during EB differentiation. Overall,

the results indicate that lack of H1c, H1d and H1e impairs the

establishment or maintenance of epigenetic changes in DNA

methylation and histone modifications that are necessary for stable

repression of pluripotent transcription factor Oct4 in differentiated

cells (Figure 5C).

Discussion

Embryonic stem cells, derived from the inner cell mass of the

blastocyst stage mammalian embryos [27,28], can self-renew

nearly indefinitely in culture and give rise to all cell types of the

three germ layers, ectoderm, mesoderm and endoderm, during

differentiation. ESCs possess distinctive transcriptional regulatory

circuits and chromatin signatures that are critical for maintaining

pluripotency and self-renewal [29,30]. Recent studies suggest that

ESCs exhibit a relatively ‘‘open’’ chromatin state, and during

differentiation, heterochromatin formation increases [2,8,31].

However, whether this ‘‘open’’ chromatin state is necessary for

pluripotency and whether the compaction of chromatin is required

for ESC differentiation remain to be addressed.

Linker histone H1 is the major chromatin architectural protein

in mediating higher order chromatin folding. H1 TKO ESCs have

Figure 5. H1 is necessary for stable repression of Oct4 pluripotency gene during embryogenesis and ESC differentiation. (A) ElevatedOct4 expression and hypomethylation of CpG sites at Oct4 promoters in H1 TKO embryos compared with littermates at E8.5. (i) qRT-PCR analysis ofmRNA expression levels of Oct4. Values are means 6 SEM, n = 5 for each genotype. Expression levels were normalized over GAPDH. *: P,0.05. (ii)Bisulfite sequencing analysis of DNA methylation status at Oct4 promoter regions. Results of two wild-type and two knockout E8.5 embryos areshown. The positions of CpG sites analyzed are depicted schematically as vertical ticks on the line. TSS: transcription start site. (iii) Percentage ofmethylated CpG sites at Oct4 promoter regions in WT and H1 TKO embryos. Statistical analysis was performed using Fisher’s exact test. ***: P,0.001;****: P,0.0001. (B) Analysis of expression and epigenetic marks at Oct4 pluripotency gene during EB differentiation in rotary suspension culture.Analyses of expression (i), DNA methylation (ii), % of mCpG (iii); and occupancy of H1 and three histone marks (iv) of Oct4 in WT, H1 TKO and RES cellsduring EB differentiation. Relative expression levels were normalized over GAPDH. Relative fold enrichment is calculated by normalizing the qChIPvalues (as described in Material and Methods) of ESCs (day 0) or EBs at each time point by that of WT ESCs (WT D0). Values are presented as mean 6S.D. *: P,0.05; **: P,0.01; ***: P,0.001. (C) Model for H1 in repression of Oct4 during ESC differentiation. ESCs have low H1 content with an relatively‘‘open’’ chromatin. During differentiation, total H1 content increases, which facilitates local chromatin compaction at Oct4 gene and contributes toestablishment and/or maintenance of epigenetic changes necessary for stable silencing of Oct4 pluripotency gene.doi:10.1371/journal.pgen.1002691.g005

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an H1/nucleosome ratio of 0.25, equivalent to 1 H1 per 4

nucleosomes, a nearly 50% reduction in total H1 levels in

comparison with WT ESCs [10]. The H1 level is especially low in

H1 TKO ESCs when compared with an H1/nucleosome ratio of

0.75,0.8 in differentiated cell types from various adult tissues

[11,15]. H1 TKO ESCs have globally decondensed chromatin

[10], offering an approachable means to examine the effect of

chromatin decondensation on ESC pluripotency and differentia-

tion. H1 TKO ESCs maintain ESC colony morphology, express

pluripotency factors (Figure 1A), propagate and self-renew

normally as wild-type ESCs, suggesting that a more ‘‘open’’

chromatin structure than normal WT ESCs does not interfere with

the ‘‘basal’’ state of ESCs, and may even promote the maintenance

of this primitive state. This prediction is consistent with the fact

that H1 TKO ESCs are easier to maintain and have sustainable

OCT4 pluripotency factor expression and robust growth even

under conditions normally promoting spontaneous differentiation,

such as culturing ESCs in the absence of LIF and feeder cells for a

prolonged period. ESCs are found to have hyperdynamic

chromatin with loosely bound major chromatin architectural

proteins, such as H1 and HP1 [6]. A more ‘‘open’’ chromatin in

H1 TKO ESCs may suggest a more dynamic chromatin structure

due to the lack of structural constraints. However, it is not clear at

present whether the remaining H1 proteins in H1 TKO ESCs

undergo a change in post-translational modifications, such as

phosphorylation, which would change the binding affinity of these

remaining H1 subtypes to chromatin [32,33]. We also note the

considerable amount of H1s remaining in these TKO ESCs, thus

further reducing H1 amount by knockout or siRNA could help

determine if a minimal level of H1 is required to permit self-

renewal of ESCs.

While a significant reduction in H1 levels does not interfere with

ESC self-renewal, it appears to clearly impair ESC differentiation.

This is manifested in static culture conditions that promote

spontaneous ESC differentiation, in a rotary suspension culture

system which induces highly reproducible and robust EB

formation and differentiation [18,34], as well as in a well defined

neural differentiation regimen. H1 TKO EBs formed in rotary

culture have a reduced level of activation of many developmental

genes and markers from all three germ layers, suggesting that the

effects of H1 depletion on differentiation and cell fate decision

broadly impact early developmental gene expression. This may

explain why only 50% of H1 TKO embryos are present at E7.5

[15]. Furthermore, H1 TKO ESCs are defective in forming

neuronal cells, glial cells, and lack formation of neural network,

which are essential for nervous system development in vivo. Total

levels of H1 increases progressively in EB formation and

differentiation, suggesting an increasingly more condensed chro-

matin state during EB differentiation in WT cultures. H1 TKO

EBs have an H1 to nucleosome ratio lower than WT ESCs. The

fact that H1 TKO ESCs cells are unable to execute normal

differentiation programs suggests that an especially low H1 level

(and the resulting more open chromatin structure [10]) impairs

ESC pluripotency and differentiation. Thus, elevated levels of the

total H1 amount as well as a more compact chromatin are not

mere consequences of differentiation processes, but a necessity to

enable it to proceed normally.

H1c, H1d, H1e and H10 are four H1 subtypes that increase

significantly during ESC differentiation. H1x, although whose

mRNA expression has been reported to increase during differen-

tiation of human ESCs and embryocarcinoma cells [35,36], is not

detected in HPLC profiles of both WT and TKO ESCs

throughout differentiation despite a 2-fold increase in mRNA

levels in TKO ESCs compared with WT ([10] and data not

shown). Thus, this more distantly related H1 subtype (H1x) is

present at a negligible level compared with the 6 somatic H1

subtypes (H1a-e and H10) in ESCs and EBs. In contrast, H1a and

H1b are abundantly present in ESCs, together accounting for one

third of total H1 content in WT ESCs. Although both H1a and

H1b increase approximately 50% in TKO ESCs upon depletion

of H1c, H1d and H1e, the levels of H1a and H1b do not increase

during EB differentiation of WT or TKO cultures. Thus, H1c,

H1d, H1e, and H10, but not H1a and H1b, are likely to be the

major contributors for the effects of H1 on ESC differentiation and

repression of pluripotency genes during ESC differentiation. In

particular, H10, a subtype highly expressed in differentiated cells

and tissues [13], progressively increases in bulk chromatin and at

the Oct4 promoter during EB differentiation and largely accounts

for the increase in total H1 levels in TKO EBs during

differentiation (Figure 4, Figure S3, and Figure S7). Thus it would

be very interesting to investigate if further deletion of H10 in the

face of H1 TKO will result in a complete inhibition of ESC

differentiation. Nevertheless, none of these four H1 subtypes alone

appears to be required for mouse ESC differentiation, because

knockout mice with deletion of one of these four H1 subtypes

develop normally [14,37], suggesting that the differentiation

defects we observed here are more likely caused by a marked

reduction of total H1 content in H1 TKO cells. Furthermore, we

show that a partial rescue of H1 content by reintroduction of H1d

into TKO cells mitigates the impairment of differentiation.

Together, we surmise that a potential threshold of H1 levels, but

not necessarily a specific H1 subtype, is required for proper ESC

differentiation.

The effects of H1 depletion on gene expression in EBs are

significant and wide-spread, drastically affecting many genes

(Figure 2C, 2D, and Figure S2C), in sharp contrast to the limited

number of genes with altered expression in H1 TKO ESCs [10]. It

is conceivable that H1 depletion in ESCs and a marked

decondensation of the chromatin pose little effects on the ‘‘basal’’

state of ESCs, but more so on impairing the capability of ESCs to

transit to differentiated cells which exhibit more compact

chromatin. Nevertheless, the influence of H1 on many develop-

mental genes in EBs could be a secondary effect resulting from the

lack of effecient repression of pluripotency gene expression, such as

Oct4 and Nanog, which associate with repressor complexes to

silence developmental genes [38]. The effects might also be caused

by misregulation of multiple key developmental genes required for

normal differentiation to proceed. It is interesting to note that 50%

of H1 TKO embryos are able to progress to mid-gestation,

suggesting that early differentiation in three germ layers in vivo is

possible for some TKO embryos [15]. Consistently, H1 TKO ES

cells are capable of forming EBs (Figure 2), albeit mostly impaired

in differentiation, and teratomas that contain a small fraction of

cells differentiated into the three germ layers (data not shown). The

impairment of ESC differentiation in vitro yet survival of some

knockout embryos to mid-gestation stage is reminiscent of several

other knockouts of ubiquitously expressed proteins that bind and

modify chromatin [8,39–41], which probably reflects more

heterogenous cell populations and conditions in vivo.

Importantly, we discovered that, compared with WT ESCs, the

H1 TKO cells fail to effectively silence the expression of

pluripotency genes Oct4 and Nanog, which are critical for

pluripotency [42,43]. We believe that this effect of H1 on

repression of Oct4 is direct because 1) Oct4 expression is higher

in H1 TKO compared with WT both in vivo in embryos and in vitro

using three differentiation schemes for ESCs and EBs, although

the degree of effects varies according to different differentiation

schemes employed; 2) reconstitution of H1d into H1 TKO ESCs

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restores the effective repression of expression and dynamic

changes in histone modifications and DNA methylation levels

during differentiation; 3) the level of H1 is cumulatively increased

at the Oct4 promoter during differentiation of WT, but not of H1

TKO, cultures. We suggest that the H1 occupancy at Oct4

promoter in ESCs could be the basal/minimal level for detection

by qChIP assay, as H1 has been found to be relatively depleted

from active promoters compared with other regions [44,45].

Interestingly, qChIP analysis showed that the association of H10 at

Oct4 promoters was significantly higher in RES cells than TKO

cells (Figure S7), suggesting that the presence of sufficient H1

proteins may facilitate H10 binding. We surmise that the

progressive increase of H1c, H1d and H1e during differentiation

and the increased H1 occupancy at Oct4 promoter lead to a

transition to a more condensed local chromatin structure necessary

for stable silencing of Oct4 during differentiation (Figure 5C).

These results together with the observation that OCT4 is present

at the promoters of several H1 subtypes in human ESCs [29,35]

suggest a potential feedback loop between OCT4 and H1 in stem

cell fate determination.

Interestingly, we found that CpG methylation of Oct4 promoter

in H1 TKO embryos is significantly reduced compared with wild-

type littermates. Although less pronounced in EB differentiation,

the effects of H1 depletion on DNA methylation at Oct4 promoter

are also apparent in day 10 EBs. This observation reinforces the

link between H1 and DNA methylation, which was initially

discovered at imprinting control regions (ICRs) of H19 and Gtl2

loci [10] and later at regulatory regions of the immunoglobin

heavy chain locus and homeobox Rhox gene cluster [46,47]. Future

studies on how DNA methylation changes at these regions in H1

TKO ESCs during differentiation will provide additional insights

on dynamic profiles of DNA methylation upon differentiation in

the face of minimal level of H1 and/or open chromatin structure.

H1 TKO EBs do not exhibit the opposite changes in the levels

of the active histone mark (H3K4me3) and the repressive histone

mark (H3K9me3) at promoters of Oct4 and Nanog that normally

occur in wild-type EBs during differentiation. Interestingly, we did

observe significant changes in the levels of histone modifications in

wild-type EBs at day 7 in rotary culture, before an increase in

DNA methylation levels occurred at Oct4 promoter. This result

reinforces the notion that DNA methylation is a slower mark to

establish compared with histone marks [48]. It is noteworthy that

the levels of DNA methylation at the Nanog promoter do not

display a difference in WT and H1 TKO embryos at day 8.5 and

are not altered during EB differentiation, suggesting that DNA

methylation is unlikely to be responsible for gene expression

changes of Nanog during this period of time.

Our results suggest a role of H1 and chromatin compaction in

epigenetic regulation of the pluripotency gene Oct4, likely

mediated through DNA methylation and histone modifications.

To our knowledge, this represents a novel mechanistic link by

which bulk chromatin compaction is directly linked to pluripo-

tency, by participating in repression of the pluripotency genes. In

ESCs, DNMT3b has been shown to interact with H1 [49]. In vitro

studies demonstrated that H1 interacts with HP1 [50,51] which

can in turn bind to SUV39H which methylates H3K9. Moreover,

H1 has been shown in vitro to stimulate the activity of PRC2

toward methylation of H3K27me3 when H1 is incorporated into

nucleosomes [52], and we have also observed interactions between

H1 and PRC2 components in ESCs (Cao, Ho, Lasater, and Fan,

unpublished observation). Therefore, we envision that during ESC

differentiation, H1 levels increase, which may facilitate the

recruitment of DNMTs, SUV39H and PRC2 to Oct4 promoter,

promoting the establishment and/or maintenance of repressive

epigenetic modifications and silencing the expression of this

pluripotency gene (Figure 5C).

In summary, we have demonstrated that loss of linker histone

subtypes H1c, H1d, and H1e impairs embryonic stem cell

differentiation. Furthermore, our results indicate that H1 contrib-

utes to silencing of pluripotency factors and participates in

mediating changes in DNA methylation and histone marks

necessary for silencing of pluripotency genes during differentiation.

Thus, modulating the levels of H1 linker histones and chromatin

compaction may potentially serve as a new strategy for regulating

stem cell pluripotency.

Materials and Methods

Embryonic stem cell cultureESC lines derived from H1 TKO and wild-type littermates were

expanded on mitotically inactivated mouse embryonic fibroblasts

feeder layers and cultured feeder-free on tissue culture-treated

dishes (Corning) pre-adsorbed with gelatin (Sigma, 0.1% solution

in ddH2O) prior to embryoid body differentiation studies. ESC

culture media consisted of Dulbecco’s modified Eagle’s medium

(DMEM) (Invitrogen) supplemented with 15% fetal bovine serum

(FBS) (Hyclone), 100 U/ml penicillin, 100 mg/ml streptomycin

and 0.25 mg/ml amphotericin (Mediatech), 2 mM L-glutamine

(Mediatech), 16 MEM non-essential amino acids (Mediatech),

0.1 mM b-mercaptoethanol (Fisher Chemical), and 103 U/ml of

leukemia inhibitory factor (LIF; ESGRO, Chemicon). Cultures

were re-fed with fresh media every other day, and passaged every

2–3 days prior to reaching 70–80% confluence. For spontaneous

differentiation studies, 26105 cells were seeded in each well of 6-

well plate at day 0 on gelatin coated plate without feeder layer,

cultured with media without LIF, and harvested at indicated time

points. Cell numbers were determined using a Multisizer 3 Coulter

Counter (Beckman).

Karyotype analysisExponentially growing ESCs were cultured in the presence of

Karyo-MAX colcemid (Gibco) for 60 minutes, washed with PBS,

trypsinized, and collected. ESCs were subsequently treated with

hypotonic solution (75 mM KCl) for 6 minutes at 37uC, fixed with

fixation solution (3 volumes Methanol, 1 volume Acetic acid),

concentrated and dropped onto an angled, humidified microscope

slide. The slide was dried and chromosomes were stained with

Hoechst dye for 1 h in the dark. Images of metaphase spread were

collected at a 606 objective on an Olympus Fluorescence

Microscope.

Rotary suspension culture and embryoid bodydifferentiation

Embryoid bodies were formed by inoculating a single-suspen-

sion of ESCs that have been passaged without feeder layers for two

generations (referred to as ‘‘day 0’’ culture) at 26105 cells/ml into

100 mm bacteriological grade polystyrene Petri dishes with 10 ml

of differentiation media (DMEM, 15% FBS, 100 U/ml penicillin,

100 mg/ml streptomycin and 0.25 mg/ml amphotericin, 2 mM L-

glutamine, 16 MEM non-essential amino acids, 0.1 mM b-

mercaptoethanol). The EB cultures were immediately placed on

rotary orbital shakers (Lab-Line Lab Rotator, Barnstead Interna-

tional) in a humidified incubator (37uC, 5% CO2) and maintained

at 40–45 rpm for the entire duration of suspension culture; rotary

speed was calibrated daily to ensure accuracy throughout. Rotary

orbital culture has been shown previously to significantly enhance

the efficiency, yield and homogeneity of EB populations compared

to static suspension culture methods [18]. Differentiation media

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was exchanged every two days by collecting EBs via gravity-

induced sedimentation in 15 ml conical tubes before aspirating

spent media, replenishing with fresh media and returning the

cultures to the rotary orbital shakers.

RNA extraction and quantitative RT–PCRTotal RNA from ESCs and embryos was extracted with Trizol

reagent (Invitrogen) and Allprep DNA/RNA Mini kit (Qiagen)

respectively according to the manufacturer’s instructions. RNA was

reverse transcribed using a SuperScript III First-strand cDNA

synthesis kit (Life Technologies). Real-time quantitative PCR

(qPCR) were performed using iQ SYBR Green Supermix with

MyIQ Single Color real-time PCR Detection System (Bio-Rad).

The following primers were used: Oct4: forward 59-GCTCA

CCCTGGGCGTTCTC-39, reverse 59-GGCCGCAGCTTACA-

CATGTTC-39; Nanog: forward 59-CCTCCAGCAGATGCAA-

GAACTC-39, reverse 59-CTTCAACCACTGGT TTTTCTG-

CC-39; Nkx2.5: forward 59-CAAGTGCTCTCCTGCTTTCC-39,

reverse 59-GGCTTTGTCCAGCTCCACT-39; alpha-MHC: for-

ward 59-GGTCCACATTCTTCA GGATTCTC-39, reverse 59-

GCGTTCCTTCTCTGACTTTCG-39; Tyrosine hydroxylase: for-

ward 59-GATTGCAGAGATTGCCTTCC-39, reverse 59-GGG-

TAGCATAGAGG CCCTTC-39; Nestin: forward 59-GCCTA-

TAGTTCAACGCCCCC-39, reverse 59-AGAC AGGCAGGGC-

TAGCAAG-39; AFP: forward 59-AAACTCGCTGGAGTGTCT-

GC-39, reverse 59-AGGTTTGACGCCATTCTCTG-39; GFAP:

forward 59-GCCACCAGT AACATGCAAGA-39, reverse 59-G-

GCGATAGTCGTTAGCTTCG; GAPDH: forward 59-TTCAC-

CACCATGGAGAAGGC-39, reverse 59-GGCATGGACTGTG-

GTCATGA-39.

PCR SuperArray analysisRNA was isolated from ESC and EB samples using QIAshred-

ders (as needed) and RNeasy Mini kits (Qiagen) according to the

manufacturer’s instructions. RNA quantity and quality were

assessed by taking absorbance measurements at 260 and 280 nm

on a NanoDrop ND1000 Spectrophotometer (Nanodrop Tech-

nologies). First strand cDNA synthesis was performed using the

RT2 First Strand Kit (SABiosciences) with 1 mg of input RNA per

well followed by real-time PCR using the Mouse Embryonic Stem

Cells PCR SuperArray and SYBR Green RT2 qPCR Master Mix

(SABiosciences), per manufacturer’s recommended protocols. First

strand synthesis and real-time PCR were performed using a

BioRad MyCycler and BioRad MyIQ real time thermal cycler,

respectively. Array results were first internally normalized to

GAPDH levels and subsequently analyzed with Genesis software

(Graz University of Technology) using log2 transformation, mean

center gene analysis, and hierarchical clustering.

Neural differentiation of ESCsESCs cultures were trypsinized with 0.25% trypsin-EDTA

solution, depleted with feeder cells, and resuspended in differen-

tiation media at 56104 cell/ml. Embryoid bodies were formed

using hanging drop method by plating 20 ml drops (1000 cells per

drop) on the inner side of the lid of 15 cm dishes. The bottom of

the 15 cm dishes were filled with sterile water and incubated for 4

days. The neural differentiation protocol for ES cells was adapted

from ES-Cult Neural differentiation protocols (StemCell Technol-

ogies, Vancouver, Canada). Briefly, four days old EBs were

collected from the hanging drops and cultured for additional 2

days in 10 cm petri dishes in the presence of 1 mM all-trans retinoic

acid. EBs were subsequently plated at 10 EBs per cm2 in tissue

culture plates, coated with poly-L-ornithin and laminin (5 mg/ml),

in NeuroCult NSC proliferation medium (StemCell Technologies)

supplemented with FGF-b 10 ng/ml. The plates were incubated

and the media was change every 2–3 days.

ImmunocytochemistryCells grown on glass cover slips were fixed with 4%

paraformaldehyde for 20 min at room temperature before

immunofluorescence staining. For immunocytochemistry, we used

the following primary antibodies: GFAP (Abcam; rabbit IgG;

1:1000), TUBB3 (Millipore; mouse IgG1; 1:50); and secondary

antibodies from Molecular Probes or Jackson Immuno Research

Laboratories: Cy3-coupled donkey anti-rabbit, Alexa Fluor 488-

coupled donkey anti-mouse antibodies. Nuclei were counter

stained with Hoechst (1:1000). Images were collected at 206and 606 on an Olympus Fluorescence Microscope.

Preparation and analysis of nuclei and histones of ESCsand EBs

mESC and EB nuclei and histones were prepared according to

protocols described previously [10,22,23]. Briefly, cultured ESCs

or EBs were harvested and nuclei were extracted using 0.5%

Nonidet P-40 in RSB (10 mM NaCl, 3 mM MgCl2, 10 mM Tris-

HCl, pH 7.5, protease inhibitors) and a Dounce homogenizer at

4uC. Released nuclei were pelleted and resuspended in RSB.

Chromatin and histone proteins were subsequently extracted as

described previously [22,23]. 50–100 mg of total histone prepara-

tions were injected into a C18 reverse phase column (Vydac) on an

AKTA UPC10 system (GE Healthcare). The effluent from the

column was monitored at 214 nm (A214), and the peaks areas were

recorded and determined with AKTA UNICORN 5.11 software.

Relative amounts of total H1s were determined by ratio of the

total A214 of all H1 peaks to half of the A214 of H2B peak. The

A214 values of the H1 and H2B peaks were adjusted to account for

the differences in the number of peptide bonds in each H1 subtype

and H2B. Fractions corresponding to the H1d/H1e peak from

HPLC analysis were collected and subjected to mass spectrometry

analysis on a Qstar XL MS/MS system (Applied Biosystems) with

electrospray ionization (ESI) as the ionization method. Analyst QS

software (Applied Biosystems) was used for data acquirement and

analysis.

Mouse embryo preparationH1c+/2H1d+/2H1e+/2 mice were set up for breeding in the

afternoon, and embryos were staged as embryonic day 0.5 (E0.5)

postcoitus at noon if a vaginal plug was found in the female in the

next morning. The female was euthanized and embryos at E8.5

were dissected from the euthanized females according to

procedures approved by Institutional Animal Care and Use

Committee. DNA and RNA were extracted from embryos using

Allprep DNA/RNA Micro kit (Qiagen) according to the

manufacturer’s instructions. Genotypes of embryos were deter-

mined by PCR assays described previously [15,53].

Quantitative chromatin immunoprecipitation (qChIP)ChIP assays were performed as described previously [10] with

modifications. Briefly, crosslinked chromatin was sheared to an

average DNA fragment size of 200 to 400 bp by sonication. 20 ml

of Dynabeads Protein G (Invitrogen) was incubated with 2 mg of

antibody for 7 hours in 4uC. After washing three times with 1 ml

PBS containing 0.5% BSA, the Dynabeads were then reacted with

40 mg of soluble chromatin overnight in 4uC. Dynabeads were

washed five times with Washing Buffer (50 mM HEPES pH 7.6,

1 mM EDTA pH 8.0, 500 mM LiCl, 0.7% Sodium Deoxycho-

late, 1% NP-40) and one time with PBS. Protein/DNA complexes

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were subsequently eluted in 100 ml Elution Buffer (50 mM Tris-Cl

pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65uC for 15 minutes,

and incubated overnight at 65uC. DNA was purified with a

Qiagen DNA Isolation column (Qiagen). The amount of each

specific DNA fragment in immunoprecipitates was determined by

real-time PCR. Triplicate PCR reactions using the iQ SYBR

Green Supermix (BioRad) were analyzed in a MyIQ Real-Time

PCR Detection System (BioRad). All samples were typically

analyzed in triplicate in two independent experiments. The

following primers were used: Oct4: forward 59-TGGGCTGAAA-

TACTGGGTTC-39, reverse 59- TTGAATGTTCGTGTGCC-

AAT-39; Nanog: forward 59-GGCATGGTGGTAGACAAGCC-

39, reverse 59-TTAGTAAGTTGGTCCATGCTTTGG-39. The

percentage of input was calculated by dividing the amount of each

specific DNA fragment in the immunoprecipitates by the amount

of DNA present in the sample before immunoprecipitation (input

DNA). The values from ChIP with control antibody (IgG) were

typically less than 5% of the ChIP values with the antibodies

against histone modifications.

AntibodiesThe following antibodies were used for Western blotting and

qChIP: anti-OCT4 (Santa Cruz sc8628), anti-GAPDH (Ambion

AM4300), anti-b ACTIN (Sigma-Aldrich A5316), anti-FLAG

(Sigma-Aldrich F3165), anti-H1 (Millipore 05-457), anti-H10

(Santa Cruz 56695), anti-H3K4me3 (Millipore 07-473), anti-

H3K9me3 (Abcam 8898), anti-H3K27me3 (Millipore 07-449),

anti-H3 (Abcam 1791) and IgG (Millipore 12-370).

Bisulfite modification, PCR amplification, and sequencinganalysis

Genomic DNA was prepared from mESCs, EBs, and embryos.

0.1 to 1 mg of DNA was treated with the Bisulfite Conversion Kit

(CpG Genome) according to the manufacturer’s manual. 1 ml of

treated DNA was used in each PCR reaction as previously

described [10]. The primers used to generate PCR products from

the bisulfite-converted DNA are specific for the converted DNA

sequence of the analyzed regions. The primer sequences were as

follows: Oct4 region1: forward 59- GATATGGGTTGAAATAT-

TGGGTTTAT-39, reverse 59-AATCCTCTCACCCCTACCT-

TAAAT-39; Oct4 region 2: forward 59-AAGGTTGAAAATG-

AAGGTTTTTTG-39, reverse 59-TCCAACCATAAAAAAAA-

TAAACACC-39; Nanog: forward 59- TTTGTAGGTGGGAT-

TAATTGTGAAT-39, reverse 59-AAAAAATTTTAAACAACA-

ACCAAAAA-39. The PCR products were subsequently cloned

using the TOPO TA Cloning kit (Invitrogen), and clones

containing the converted DNA inserts were picked and sequenced.

DNA sequences were analyzed with BiQ analyzer [54].

Generation of H1d rescue (RES) cell linesThe H1d overexpression plasmid was constructed by cloning a

5 Kb fragment encompassing H1d coding region (with an

insertion of FLAG tag at N-terminus) and proximal regulatory

sequences into a vector containing a Blasticidin resistant gene.

20 mg of plasmid DNA was transfected into 26107 H1 TKO ESCs

as described before [14], and 96 cell clones resistant to blasticidin

were picked and analyzed by Western blotting using an anti-

FLAG antibody (Sigma-Aldrich). Two cell lines with the highest

levels of H1d were selected as RES cell lines for further analysis.

Supporting Information

Figure S1 Chromosome spreads of WT and H1 TKO ESCs.

(TIF)

Figure S2 Gene expression analysis of ESCs and EBs formed in

rotary suspension culture. (A) qRT-PCR analysis of expression

levels of Nkx2.5 and a-MHC in WT and H1 TKO cells during EB

differentiation. Expression levels were normalized over GAPDH.

(B) List of genes that displayed more than two-fold differences

(P,0.05) in expression shown in Figure 2Di, 2Dii and 2Diii,

respectively. (C) Scatter plot analysis comparing the degree of

changes in gene expression in WT and H1 TKO cells during EB

differentiation. X-axes and y- axes are delta delta CTs.

(TIF)

Figure S3 Analysis of total H1 and H10 levels during EB

differentiation. 2 mg histone proteins were analyzed with immu-

noblotting with antibodies indicated. The bottom panel of

Western blotting with anti-H3 antibody demonstrates equal

loading of proteins in each lane.

(TIF)

Figure S4 Increased expression of Nanog by H1 depletion in

embryos. (A) qRT-PCR analysis of E8.5 embryos indicating the

higher levels of Nanog expression in H1 TKO embryos compared

with WT. Values are means 6 SEM, n = 5 for each genotype.

Expression levels were normalized over GAPDH. *: P,0.05. (B)

DNA methylation status of promoter regions of Nanog in E8.5

embryos analyzed by bisulfite sequencing. (C) Percentage of CpG

methylation calculated from results in (B).

(TIF)

Figure S5 Generation and characterization of RES ESC lines.

(A) Representative Western blotting analysis of ‘‘rescue’’ clones.

Immunoblotting with anti-b-ACTIN antibody indicates equal

loading of whole cell lysates. (B) Reverse phase HPLC analysis of a

RES cell line with high levels of H1d expression. (C) Chromosome

spread of the RES cell shown in B). (D) Hematoxylin and eosin

staining of sections of day 10 EBs generated from RES cells in

rotary suspension culture. Scale bar: 100 mm. (E) qRT-PCR

analysis of differentiation markers in RES cells during EB

differentiation. Expression levels were normalized over GAPDH.

(TIF)

Figure S6 Analysis of expression and epigenetic marks at Nanog

promoter. (A) qRT-PCR analysis of Nanog expression in ESCs and

day 10 EBs. Expression levels were normalized over GAPDH. (B)

DNA methylation status of Nanog promoter in mouse embryonic

fibroblasts (MEFs) (left) or in ESCs (day 0) and day 10 EBs (right).

(C) qChIP analysis of H1, H3K4me3, H3K9me3 and H3K27me3

levels at Nanog promoters in ESCs (day 0) and day 10 EBs. Data

were normalized as described in Figure 5Biv. *: P,0.05;

**: P,0.01.

(TIF)

Figure S7 qChIP Analysis of H10 occupancy at Oct4 promoter

during EB differentiation. Data were normalized as described in

Figure 5Biv. *: P,0.05; **: P,0.01; ***: P,0.001.

(TIF)

Acknowledgments

We thank colleagues and lab members for critical reading of this

manuscript, and the anonymous reviewers for their comments. We thank

Samantha Lasater for editorial assistance.

Author Contributions

Conceived and designed the experiments: YF TCM. Performed the

experiments: YZ MC SP KC BK P-YH MM DTB CP YF. Analyzed the

data: YZ MC SP KC BK MM TCM YF. Contributed reagents/materials/

analysis tools: YF TCM. Wrote the paper: YF TCM SP KC.

Loss of H1 Impairs Stem Cell Differentiation

PLoS Genetics | www.plosgenetics.org 13 May 2012 | Volume 8 | Issue 5 | e1002691

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PLoS Genetics | www.plosgenetics.org 14 May 2012 | Volume 8 | Issue 5 | e1002691